Studies on the Effect of Fructose Diphosphatase on Phosphofructokinase

Kosaku Uyeda,Lynne J Luby

doi:10.1016/s0021-9258(19)42455-1

Abstract

Abstract Chicken liver fructose diphosphatase has been prepared in a homogeneous form. The molecular weights of the enzyme and the dissociated enzyme were determined to be 142,000 and 38,000, respectively, by a high speed sedimentation equilibrium technique and by acrylamide gel electrophoresis in sodium dodecyl sulfate. The effect of fructose diphosphatase on phosphofructokinase was investigated using purified preparations of these enzymes from chicken liver and rabbit muscle. Fructose diphosphatase enhances ATP inhibition of phosphofructokinase. However, fructose diphosphatase which had been treated with homocystine, CoA, p-chloromercuribenzoate, or iodoacetamide loses this ability to enhance the ATP inhibition of phosphofructokinase. Among various proteins examined, only fructose diphosphatase showed this effect. Phosphofructokinase shows sigmoidal kinetics with respect to fructose-6-P, but in the presence of fructose diphosphatase the sigmoidicity is increased significantly and s0.5 for fructose-6-P is increased with increasing fructose diphosphatase concentration. ATP inhibition of phosphofructokinase is less at pH 8 than at pH 7.4; however, fructose diphosphatase enhances this inhibition at pH 8. Fructose diphosphatase also increases the inhibition of phosphofructokinase by 3-P-glycerate and citrate. The molar ratios of fructose diphosphatase to phosphofructokinase which give 50% inhibition varies from 100 to 400 depending upon the sources of the enzymes. These kinetic studies suggest that fructose diphosphatase may cause conformational changes in phosphofructokinase. In order to examine a possible interaction between these enzymes, the effect of fructose diphosphatase on the fluorescence of phosphofructokinase-anilinonaphthol sulfonate complex was investigated. Adenine nucleotides quench the fluorescence of the complex, and fructose diphosphatase enhances the quenching of the fluorescence further. Homocysteine or CoA derivatives of fructose diphosphatases, however, showed no effect on the fluorescence. These fluorescence data are consistent with the kinetic results and suggest that fructose diphosphatase enhances allosteric effects of phosphofructokinase. A possible significance of these observations in the regulation of the futile cycle and also coordinated control of glycolysis and gluconeogenesis is discussed.

Highlights

Chicken liver fructose diphosphatase has been prepared in a homogeneous form
ATP inhibition of phosphofructokinase is less at pH 8 than at pH 7.4; fructose diphosphatase enhances this inhibition at pH 8
We have reported in a preliminary communication [14] that fructose 1,&diphosphatase in, vilro enhances allosteric inhibition of phosphofructokinase by ATP or 3-P-glycerate, by removal of fructose-l,6-1’2, and by inducing conformational changes in phosphofructokinase

Summary

Methods

Fructose diphosphatase activity was determined either at pH 9.6 or 7.0. The assav at nH 9.6 was nerformed as follows. The reaction mixture coitained in a final volume of 1 ml: 100 mM glycine at pH 9.6; fructose-1,6-P2,O.l mM; NADP, 0.2 mM; MgCl2, 1 mM; El>TA, 0.2 mM; phosphoglucose isomerase, 2 units; and glucose 6.phosphat,e dehydrogenase, 2 units. The reaction was initiated by addition of fructose diphosphatase and the rate of the formation of NADPH was measured at, 28” wit,h a Gilford recording spectrophotometer. The pH 7 (“neutral”) assay contained the same reagents as above except Tris-Cl at pH 7.0 was used as a buffer. One unit of fructose diphosphatase activity is defined as that amount of the enzyme which catalyzes the formation of 1 pmole of fructose-6-P per min

Results

Discussion

Conclusion